The overall goal of the proposed effort is to use the principles of intermolecular forces to design improved cationic amphiphile/DNA supramolecular assemblies for gene delivery. Specifically, we will design, synthesize, and evaluate new charge-reversal amphiphiles for gene delivery. These functional amphiphiles undergo an electrostatic transition from cationic to anionic in cells to release DNA from the supramolecular assembly. We hypothesize that this change in electrostatic interactions with DNA will translate to enhanced gene transfection efficiency. A detailed mechanistic investigation is proposed which entails the following three specific aims for this four-year proposal: Aim 1. Determine the key molecular characteristics of the amphiphile required for 1) DNA binding and release of DNA from the supramolecular assembly and 2) destabilization of lipid bilayers. Aim 2. Characterize the charge-reversal amphiphiles and amphiphile/DNA supramolecular assemblies. Aim 3. Evaluate functional interactions of charge-reversal amphiphile/DNA supramolecular assemblies with cells in vitro and deliver the p53 gene to breast cancer cells. Completion of these aims will afford: (1) the identification of one or more charge-reversal amphiphiles that can deliver DNA to cells;(2) the mechanism of nucleic acid delivery with these charge-reversal amphiphiles;(3) an understanding of the advantages and limitations of these amphiphiles for gene delivery in a given cell type;(4) the demonstration of a new approach that is a conceptual departure from the current gene delivery vectors;(5) delivery of the p53 gene to breast cancer cells;and (6) potentially identify the nature of the rate- limiting step in gene delivery. Breast cancer is one disease that would benefit from improved or alternative treatment options. Today, there is no standard of care for metastatic breast cancer;all of the first-line combination therapies are regarded as equally efficacious at about a 60% response rate. Thus, we are focusing on the delivery of the tumor suppressor gene (p53) for the treatment of breast cancer.